Research Article| Volume 167, ISSUE 3, P513-518, December 2022

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DNA methylation of the immediate upstream region of BRCA1 major transcription start sites is an independent favorable prognostic factor in patients with high-grade serous ovarian cancer

Published:October 14, 2022DOI:


      • A pyrosequencing primer set was developed to measure the methylation level of an immediate upstream region of BRCA1.
      • BRCA1 methylation was observed in 21.5% of high-grade serous ovarian cancers.
      • BRCA1 methylation was a prognostic factor for progression-free survival in patients with high-grade serous ovarian cancer.



      To establish a quantitative method to evaluate the DNA methylation level of an immediate upstream region of major BRCA1 transcriptional start sites (TSSs), and to investigate whether methylation of the region is a prognostic factor in high-grade serous ovarian cancer patients after neoadjuvant chemotherapy.


      Ninety-two FFPE samples of advanced high-grade serous ovarian cancers after neoadjuvant chemotherapy between 2011 and 2018 were used for mutation and methylation analysis. DNA methylation levels were assessed by pyrosequencing and DNA methylation microarray. An association between methylation level (or a mutation) and progression-free survival was assessed by Kaplan-Meier analysis.


      Major BRCA1 transcripts and CpG sites immediately upstream of their TSSs were identified, and a pyrosequencing method was developed. BRCA1 methylation, BRCA1/2 mutations, and a RAD51C mutation were detected in 17/79 (21.5%), 17/92 (18.5%), and 1/92 (1.1%) high-grade serious ovarian cancer samples. In univariate analysis, BRCA1 methylation and no residual tumor were associated with progression-free survival (BRCA1 methylation: P = 0.025, no residual tumor: P = 0.0026). Multivariate analysis showed that both BRCA1 methylation (P = 0.038, HR = 0.47, 95% CI: 0.21–0.96) and no residual tumor (P = 0.012, HR = 0.49, 95% CI: 0.28–0.85) were significant favorable prognostic factors.


      A quantitative method to estimate the methylation level of the immediate upstream region of major BRCA1 TSSs was established. Methylation of the region of was an independent favorable prognostic factor in high-grade serous ovarian cancer patients.


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      1. Cancer Genome Atlas Research Network, Integrated genomic analyses of ovarian carcinoma.
        Nature. 2011; 474: 609-615
        • Tan D.S.
        • Rothermundt C.
        • Thomas K.
        • Bancroft E.
        • Eeles R.
        • Shanley S.
        • et al.
        “BRCAness” syndrome in ovarian cancer: a case-control study describing the clinical features and outcome of patients with epithelial ovarian cancer associated with BRCA1 and BRCA2 mutations.
        J. Clin. Oncol. Off. J. Am. Soc. Clin. Oncol. 2008; 26: 5530-5536
        • Cunningham J.M.
        • Cicek M.S.
        • Larson N.B.
        • Davila J.
        • Wang C.
        • Larson M.C.
        • et al.
        Clinical characteristics of ovarian cancer classified by BRCA1, BRCA2, and RAD51C status.
        Sci. Rep. 2014; 4: 4026
        • Yang D.
        • Khan S.
        • Sun Y.
        • Hess K.
        • Shmulevich I.
        • Sood A.K.
        • et al.
        Association of BRCA1 and BRCA2 mutations with survival, chemotherapy sensitivity, and gene mutator phenotype in patients with ovarian cancer.
        JAMA. 2011; 306: 1557-1565
        • Alsop K.
        • Fereday S.
        • Meldrum C.
        • deFazio A.
        • Emmanuel C.
        • George J.
        • et al.
        BRCA mutation frequency and patterns of treatment response in BRCA mutation-positive women with ovarian cancer: a report from the Australian ovarian cancer study group.
        J. Clin. Oncol. Off. J. Am. Soc. Clin. Oncol. 2012; 30: 2654-2663
        • Ledermann J.
        • Harter P.
        • Gourley C.
        • Friedlander M.
        • Vergote I.
        • Rustin G.
        • et al.
        Olaparib maintenance therapy in patients with platinum-sensitive relapsed serous ovarian cancer: a preplanned retrospective analysis of outcomes by BRCA status in a randomised phase 2 trial.
        Lancet Oncol. 2014; 15: 852-861
        • Swisher E.M.
        • Lin K.K.
        • Oza A.M.
        • Scott C.L.
        • Giordano H.
        • Sun J.
        • et al.
        Rucaparib in relapsed, platinum-sensitive high-grade ovarian carcinoma (ARIEL2 part 1): an international, multicentre, open-label, phase 2 trial.
        Lancet Oncol. 2017; 18: 75-87
        • Mirza M.R.
        • Monk B.J.
        • Herrstedt J.
        • Oza A.M.
        • Mahner S.
        • Redondo A.
        • et al.
        Niraparib maintenance therapy in platinum-sensitive, recurrent ovarian cancer.
        N. Engl. J. Med. 2016; 375: 2154-2164
        • Swisher E.M.
        • Kwan T.T.
        • Oza A.M.
        • Tinker A.V.
        • Ray-Coquard I.
        • Oaknin A.
        • et al.
        Molecular and clinical determinants of response and resistance to rucaparib for recurrent ovarian cancer treatment in ARIEL2 (parts 1 and 2).
        Nat. Commun. 2021; 12: 2487
        • Lin J.C.
        • Jeong S.
        • Liang G.
        • Takai D.
        • Fatemi M.
        • Tsai Y.C.
        • et al.
        Role of nucleosomal occupancy in the epigenetic silencing of the MLH1 CpG island.
        Cancer Cell. 2007; 12: 432-444
        • Yamashita R.
        • Sathira N.P.
        • Kanai A.
        • Tanimoto K.
        • Arauchi T.
        • Tanaka Y.
        • et al.
        Genome-wide characterization of transcriptional start sites in humans by integrative transcriptome analysis.
        Genome Res. 2011; 21: 775-789
        • Ghandi M.
        • Huang F.W.
        • Jané-Valbuena J.
        • Kryukov G.V.
        • Lo C.C.
        • McDonald 3rd, E.R.
        • et al.
        Next-generation characterization of the cancer cell line encyclopedia.
        Nature. 2019; 569: 503-508
        • Esteller M.
        • Silva J.M.
        • Dominguez G.
        • Bonilla F.
        • Matias-Guiu X.
        • Lerma E.
        • et al.
        Promoter hypermethylation and BRCA1 inactivation in sporadic breast and ovarian tumors.
        J. Natl. Cancer Inst. 2000; 92: 564-569
        • Ruscito I.
        • Dimitrova D.
        • Vasconcelos I.
        • Gellhaus K.
        • Schwachula T.
        • Bellati F.
        • et al.
        BRCA1 gene promoter methylation status in high-grade serous ovarian cancer patients--a study of the tumour Bank ovarian cancer (TOC) and ovarian cancer diagnosis consortium (OVCAD).
        Eur. J. Cancer. 2014; 50: 2090-2098
        • Hodgson D.R.
        • Dougherty B.A.
        • Lai Z.
        • Fielding A.
        • Grinsted L.
        • Spencer S.
        • et al.
        Candidate biomarkers of PARP inhibitor sensitivity in ovarian cancer beyond the BRCA genes.
        Br. J. Cancer. 2018; 119: 1401-1409
        • Thakur S.
        • Croce C.M.
        Positive regulation of the BRCA1 promoter.
        J. Biol. Chem. 1999; 274: 8837-8843
        • Atlas E.
        • Stramwasser M.
        • Mueller C.R.
        A CREB site in the BRCA1 proximal promoter acts as a constitutive transcriptional element.
        Oncogene. 2001; 20: 7110-7114
        • Kalachand R.D.
        • Stordal B.
        • Madden S.
        • Chandler B.
        • Cunningham J.
        • Goode E.L.
        • et al.
        BRCA1 promoter methylation and clinical outcomes in ovarian cancer: an individual patient data meta-analysis.
        J. Natl. Cancer Inst. 2020; 112: 1190-1203
        • Fujiwara K.
        • Hasegawa K.
        • Nagao S.
        Landscape of systemic therapy for ovarian cancer in 2019: primary therapy.
        Cancer. 2019; 125: 4582-4586
        • Ebata T.
        • Yamashita S.
        • Takeshima H.
        • Yoshida H.
        • Kawata Y.
        • Kino N.
        • et al.
        DNA methylation marker to estimate ovarian cancer cell fraction.
        Med. Oncol. 2022; 39: 78
        • Yamaguchi T.
        • Mukai H.
        • Yamashita S.
        • Fujii S.
        • Ushijima T.
        Comprehensive DNA methylation and extensive mutation analyses of HER2-positive breast cancer.
        Oncology. 2015; 88: 377-384
        • Ueda S.
        • Yamashita S.
        • Watanabe S.I.
        • Wakabayashi M.
        • Motoi N.
        • Noguchi M.
        • et al.
        Influence of degree of DNA degradation in formalin-fixed and paraffin-embedded tissue samples on accuracy of genome-wide DNA methylation analysis.
        Epigenomics. 2021; 13: 565-576
        • Sahnane N.
        • Carnevali I.
        • Formenti G.
        • Casarin J.
        • Facchi S.
        • Bombelli R.
        • et al.
        BRCA methylation testing identifies a subset of ovarian carcinomas without germline variants that can benefit from PARP inhibitor.
        Int. J. Mol. Sci. 2020; 21
        • Cohen P.A.
        • Powell A.
        • Böhm S.
        • Gilks C.B.
        • Stewart C.J.R.
        • Meniawy T.M.
        • et al.
        Pathological chemotherapy response score is prognostic in tubo-ovarian high-grade serous carcinoma: a systematic review and meta-analysis of individual patient data.
        Gynecol. Oncol. 2019; 154: 441-448